virus-specific T-cell clones in vivo

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glutinin or neuraminidase, can receive help from TH clones specific for any of ..... by hemagglutination inhibition and neuraminidase inhibition tests, respectively.
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 4446-4450, June 1988 Immunology

Differential ability of B cells specific for external vs. internal influenza virus proteins to respond to help from influenza virus-specific T-cell clones in vivo PEGGY A. SCHERLE AND WALTER GERHARD The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104

Communicated by Hilary Koprowski, February 22, 1988

ABSTRACT When a helper T-cell (TH) clone specific for the hemagglutinin, neuraminidase, matrix protein, or nucleoprotein of influenza strain A/PR/8/34 is adoptively transferred to athymic mice 1 day after virus infection the anti-viral antibody response of the mouse is enhanced. This response is directed predominantly to the hemagglutinin and requires associative T-cell-B-cell interactions. Delaying transfer of the TH clone has three consequences: (a) the onset of the antihemagglutinin antibody response is delayed; (it) the titer of the anti-hemagglutinin response is reduced; and (ifi) the titer of the antibody in the response against the internal proteins, matrix protein and nucleoprotein, is enhanced upon transfer of matrix protein- or nucleoprotein-speciflic, but not hemagglutinin- or neuraminidase-specific, TH clones. Thus, there is a hierarchy of help: B cells recognizing viral surface components, hemagglutinin or neuraminidase, can receive help from TH clones specific for any of the major structural viral proteins. In contrast, B cells responding to internal viral components, matrix protein or nucleoprotein, are restricted to receiving help almost exclusively from TH clones with the same protein specificity. These observations suggest that, upon B-cell surface immunoglobulin-antigen interaction and uptake of intact virus, B cells specific for viral surface proteins process and present all major structural viral antigens, enabling the B cells to interact with TH clones specific for any virion protein. B cells recognizing internal viral components, which may be accessible to interaction with B-cell immunoglobulin receptors mainly as free proteins, would present only the protein for which they are specific and, thereby, receive help only from the TH clones of the same protein specificity.

The types of T-cell-B-cell interactions and the mechanisms

by which they occur have in general been defined with hapten-carrier systems. Whether these same interactions occur in response to a complex antigen, such as a virus, is unclear. The influenza virus, in particular, can potentially adsorb to all cells by initially binding to sialic acid residues on cell-surface proteins (18). It can also infect many cell types, including lymphocytes (19). Further, because there is biosynthesis of new virus particles as well as the release of viral proteins when the infected cells lyse, the virus as an antigen

is in a continually dynamic state. We have, therefore, been studying the T-cell-B-cell interactions that occur in the response to influenza virus infection in vivo. We have shown (20) that TH clones specific for the hemagglutinin (HA), nucleoprotein (NP), or matrix protein (M) provide effective help for an antiviral antibody response upon adoptive transfer to nu/nu mice 1 day after virus infection. This response is directed almost exclusively to the HA and requires a cognate T-cell-B-cell interaction whether the determinants recognized by the T cell and B cell are located on the same virus protein or on different proteins within the same virus particle. In the present study, we extend these findings and show that delaying transfer of TH clones specific for the internal viral proteins M or NP, or for the surface glycoproteins HA or neuraminidase (NA), until 3 days after virus infection delays the onset and reduces the titer of the anti-HA antibodies in the response. Further, delayed transfer of the TH specific for M or NP, but not HA or NA, significantly enhances the antibody response to the internal proteins. Finally, although the B-cell response to the surface proteins HA and NA is supported equivalently by TH specific for internal or external viral proteins, generation of an antibody response to the internal proteins appears to require that the B cell and the TH recognize determinants on the same virus protein. A model of the T-cell-B-cell interactions occurring in response to influenza virus, which accommodates these findings, is proposed.

For a resting B cell to become activated and produce antibodies, a second signal, beyond the occupancy of its surface immunoglobulin receptor by antigen, is required (1, 2). This signal is in most cases provided by helper T cells (TH). Two types of T-cell help have been described (3, 4). Cognate help is major histocompatibility complex-restricted and requires that the antigenic determinants recognized by the B cell and by the T cell be covalently linked (5-8). This type of help is thought to occur through a direct interaction

MATERIALS AND METHODS Viruses and Viral Proteins. The A/PR/8/34 (HiNl) (or PR8) virus was grown and purified, and the titer was determined as described (21). HA and viral cores were prepared by bromelain treatment of the virus (22). M and NP were prepared and their purity was assessed as described

between the TH and B cells and to result in the transduction of a signal to the B cell in the form of locally released factors and/or crosslinking of surface molecules. This signal enables the B cell to go on to proliferate and differentiate into an antibody-secreting B-cell clone (9-13). Bystander help, in contrast to cognate help, is thought to occur through the release of T-cell-derived factors that act nonspecifically on activated B cells (14-17). No direct link between the antigenic determinants recognized by the T cell and the B cell is, therefore, required.

(23). Virus Infection in Vivo. BALB/cbyJ female mice, 5-7 weeks of age (The Jackson Laboratory), and BALB/c nu/lnu female mice, 5-8 weeks of age (Harlan Sprague Dawley, Indianapolis, IN), were infected with an aerosol preparation of PR8 virus as described (20). Nu/nu mice were reconsti-

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Abbreviations: HA, hemagglutinin; M, matrix; NA, neuraminidase; NP, nucleoprotein; PR8, A/Puerto Rico/8/34 (HlNl); TH, helper T cell(s).

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Proc. Natl. Acad. Sci. USA 85 (1988)

tuted by intravenous injection of 1-2 x 106 cloned T cells 1, 3, 5, 7, or 9 days later. Serum was collected at various intervals thereafter by retroorbital bleeding. TH Clones. T-cell clones, V1.2 (HA-specific), TL1/3.1 (NP-specific), and T2.5-26 (M-specific), were isolated and maintained in vitro as described (20, 24). The TH clone 5.1-7 was isolated from the popliteal lymph nodes of a BALB/c mouse that had been primed by aerosol infection and given a booster injection of PR8 (100 HA units) in complete Freund's adjuvant into the footpad =30 days later. The clone recognizes a determinant on the NA in the context of the I-Ed class II molecule. For reconstitution of nude mice at each time point after injection, TH at the same stage after antigen stimulation (10-15 days) were used and were separated from dead cells and debris by centrifugation onto a cushion of Ficoll/Hypaque immediately before transfer. Hemagglutination Inhibition and NA Inhibition Assays. The hemagglutination inhibition assay was performed in 96-well polystyrene microtiter plates as described (25). The NA inhibition test was performed as described by Russ et al. (26) except that the serum and virus were incubated overnight with fetuin (GIBCO). The reassortant virus J1 (H3N1) (27) was used to eliminate the possibility of steric inhibition of NA activity by anti-HA antibodies. The optical density of the reaction mixture was measured on a Bio-Tek (Burlington, VT) microtiter plate reader (model EL309) at a test wavelength of 570 nm and a reference wavelength of 750 nm. The anti-NA titer was determined as the dilution of serum that gave 50% inhibition of NA activity. RIA and ELISA. The level of serum anti-M and anti-NP antibodies was determined by radioimmunoassay (RIA) as described (28). Briefly, wells of a 96-well polyvinyl microtiter plate were coated with purified M (-250 ng per well) or NP (-200 ng per well) and developed with the iodinated rat anti-mouse K-light-chain constant region (CK) monoclonal antibody, antibody 187.1 (29). The antibody response to core proteins was measured in an ELISA. The plates were coated with viral cores (-300 ng per well), and the assay was performed exactly as the RIA except that the plates were developed with biotinylated rat anti-mouse K-light-chain constant-region monoclonal antibody (antibody 187.1), followed by avidin-peroxidase (Sigma) (0.2 /g/ml) and the substrate tetramethylbenzidine (Sigma) (100 ,g/ml) with 0.015% hydrogen peroxide in ELISA buffer (0.1 M NaOAc adjusted with citric acid to pH 6.0) as described (30). The reaction was stopped after =20 min by adding 2 M H2SO4. All reagents were added in a volume of 25 ,ul per well. The optical density in the ELISA was measured on a Bio-Tek microtiter I--

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plate reader (model EL309) at a test wavelength of 450 nm and a reference wavelength of 750 nm. RESULTS We had observed (20) that when individual TH clones were adoptively transferred to T-cell-deficient mice (BALB/c nu/nu) 1 day after virus infection, the mice could mount an anti-viral antibody response. Interestingly, however, this antibody response was directed predominantly to the major viral surface glycoprotein HA, whether the adoptively transferred TH clone was specific for the HA or for NP or M, the internal viral proteins. This was in contrast to the antibody response of immunocompetent BALB/c mice, which was directed to both external and internal viral proteins. To account for the failure of adoptively transferred TH clones to help B cells respond to M or NP, we considered the possibility that B cells specific for internal viral proteins might receive antigen-specific stimuli that would enable them to respond to T-cell help at a time when the transferred TH clones had become inactive. For instance, the main form of antigen recognized by these B cells might be free internal viral proteins released when virus-infected cells lysed later in the course of the virus infection. To test this proposal, TH clones were transferred to nu/nu mice at various times after aerosol infection, and the sera from the animals were tested for titer and specificity of the anti-viral antibodies produced. Delayed transfer of the TH had two main consequences (Fig. 1). First, transfer of TH clones specific for M, NP, HA, or NA (Fig. 1 A-D, respectively) 3 days after infection concomitantly delayed the onset and reduced the titer of the anti-HA antibodies in the response. Nude mice that received TH 3 days after infection had detectable levels of anti-HA antibodies 2-4 days later than mice that received the same TH clones 1 day after infection. Further, the magnitude of the response supported by each TH clone was lower by a factor of 2-4 when transfer was delayed. Additionally, as exemplified by transfer of the M-specific clone (Fig. LA), transfer delayed for more than 3 days after infection further retarded the onset and decreased the magnitude of the anti-HA response; such that, by 9 days after infection, transferred TH failed to support a response above that of unreconstituted nude mice. These results cannot be attributed to a decrease in viral antigens over the 9-day period, since virus titers in the lungs of the nude mice increased during the first 5 days after infection and remained at consistently high levels thereafter (data not shown). The second consequence of delayed transfer concerned the antibody response to the major viral core proteins M and NP.

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FIG. 1. Effect of delayed transfer of TH clones on the antibody response to the HA and viral core proteins. The serum anti-HA titer of nude mice receiving the M (A), NP (B), HA (C), or NA (D) specific TH clones 1 (o), 3 (A), 7 (o), or 9 (*) days after infection was determined by a hemagglutination inhibition (HI) assay. Anti-core antibodies in serum from nude mice after adoptive transfer of a TH clone specific for M (E), for NP (F), for HA (G), or for NA (H) 1 (o), 3 (A), or 5 (o) days after infection was determined by ELISA. The data shown represent the optical density obtained with a 1:100 dilution of serum. The anti-core response of unreconstituted nude mice (*) at the same dilution is shown in E. All assays were performed on a pool of sera from four or five mice and represent the mean antibody titers obtained from two or three individual experiments.

Immunology: Scherle and Gerhard

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Proc. Natl. Acad Sci. USA 85 (1988)

Fig. 1 E-H shows the anti-core antibody titers in nude mice reconstituted 1 or 3 days after infection with an M-, NP-, HAor NA-specific TH clone, respectively. As reported (20), transfer of M- and NP-specific TH clones 1 day after infection resulted in a minimal and inconsistently detectable antibody response to viral core proteins. However, transfer of these TH clones 3 days after infection reproducibly enhanced this response to clearly detectable levels. This enhancement could no longer be detected when the transfer of the TH was delayed until 5 days or more after infection (Fig. 1E and data not shown). By contrast, neither the HA- nor the NA-specific TH clone, whether transferred early or late after infection, supported an antibody response to viral core proteins. The specificity of the anti-viral antibody response in the reconstituted nude mice was further examined by quantitating antibody titers to the individual core proteins, M, and NP. Fig. 2 shows the maximal titers obtained from nude mice receiving the TH 3 days after infection relative to unrecon-, stituted nude mice controls. It can be seen that B cells specific for HA or NA, the surface glycoproteins, are helped equivalently by TH clones, specific for M, NP, HA, or NA. In contrast, the M- and NP-specific B cells receive help preferentially from TH clones of the same protein specificity. Again, the HA-specific TH clone is unable to significantly enhance either the M- or the NP-specific antibody responses. It is interesting, also, that normal BALB/c mice failed to mount a significant anti-M antibody response in contrast to the roughly 8-fold stronger anti-M response of nude mice after reconstitution with the M-specific TH clone.

DISCUSSION Here and in a previous study (20), we have made use of adoptive transfer of individual TH clones to influenza virusinfected athymic BALB/c mice to characterize the T-cell-Bcell interactions that are operative in the anti-viral antibody response. The basic characteristics of the experimental

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